The invention relates to a novel homogeneous method of detecting and/or determining the phosphorylating activity of a biological material towards a substrate containing tyrosine and/or serine and/or threonine, and to a kit for carrying out this method.
The phosphorylation of biological molecules, such as peptides or proteins, by kinases is a major biological mechanism for regulating cell metabolism.
The majority of enzymes which possess a phosphorylating activity have a very high Km (Michaelis constant) (generally of between 10xe2x88x923 and 10xe2x88x925 M) and a very low conversion yield (between 5% and 0.001% of the active sites of the substrate are phosphorylated).
Under these conditions, it is only possible to detect the phosphorylation of a substrate if the active sites are present in large excess during the reaction. This large excess of active sites can be obtained either by using high concentrations of substrate (if it only has a few active sites) or by choosing a substrate which possesses a large number of phosphorylation sites.
To date, the mechanisms of phosphorylation have generally been studied by radioactive or enzymatic heterogeneous methods of detection.
In methods of this type, the phosphorylation of the substrate fixed to a solid phase is detected either by measuring the incorporation of 32P into the enzyme substrate or by using a labeled antibody (isotopic, enzymatic or fluorescent tracer) directed against the phosphorylation site.
This type of assay makes it possible to fix a large amount of substrate to the solid phase and hence to detect the phosphorylation even when the substrate possesses only a small number of active sites, but it nevertheless has major disadvantages, namely:
the frequent use of isotopic markers,
the need for separation processes between the different steps of the assay in order to remove the excess reagents, and
the need to control the substrate xe2x80x9ccapturexe2x80x9d processes (for example when using a plate carrying avidin with a biotin substrate).
In the case of a homogeneous method, it is often necessary for the concentration of the substrate to be high in order to generate a sufficient amount of phosphorylated substrate to detect. It then becomes difficult to capture all the substrate as this would require a large amount of reagent, which, if the reagent is fluorescent, has the disadvantage of generating a high specific background noise.
It has now been found that the phosphorylation of a substrate can be detected by means of a homogeneous method using a luminescent carrier molecule to which a plurality of substrates are covalently coupled. After the enzymatic phosphorylation reaction, the amount of phosphorylated substrate is disclosed by measuring the signal emitted by the luminescent carrier molecule and generated by energy transfer from a specific receptor for the phosphorylated substrate labeled with a luminescent molecule.
This method is particularly useful for measuring the phosphorylation of molecules of biological interest, for example peptides, polypeptides, proteins or nucleotides, in natural or pathological processes, or in synthetic processes such as the synthesis of nucleic acids or proteins.